Abstract The studies in Project 1 will center on elucidating structural determinants for a second, non-catalytic function of HIV-1 integrase (IN) during virion maturation. While an essential role of IN for integration of viral cDNA into human chromosome during early steps of HIV-1 infection has been long established and successfully exploited as a therapeutic target, the recent studies with allosteric IN inhibitors (ALLINIs) by HIVE investigators coupled with earlier site-directed mutagenesis experiments have suggested that IN plays an active role in viral particle maturation. During the past funding period our studies with ALLINIs, a new class of very promising antiviral compounds, which are currently in clinical trials, have uncovered that these inhibitors induce hyper or aberrant multimerization of HIV-1 IN and yield eccentric, non-infectious virions where the ribonucleoprotein complexes (RNPs) are mislocalized outside of the translucent capsid cores. Subsequent collaborative efforts by HIVE and CRNA investigators have revealed that IN binds the viral RNA genome to ensure correct localization of RNPs within the protective capsid core, whereas ALLINI treatments impair IN-RNA interactions and result in the eccentric core phenotype. These seminal findings will now be extended to elucidate key structural interactions that underlie these biological events. In particular, our experiments will take advantage of cutting-edge technologies available within the HIVE center as well as utilize complementary expertise of CRNA and PCHPI scientists to accomplish the following aims. Aim 1 will determine the cryo-EM structure of IN bound to a cognate RNA element; aim 2 will dissect complementary interactions of IN and nucleocapsid with viral RNA using single molecule fluorescence and SHAPE; aim 3 will examine the significance of IN-RNA interactions in non-primate lentiviruses and other retroviruses using CLIP-seq and biochemical approaches; aim 4 will investigate the structural basis for ALLINI induced hyper-multimerization of full length wild type IN using solid state NMR and free energy calculations; and aim 5 will generate mesoscale models of correctly matured and ALLINI treated eccentric virus particles. These highly innovative experiments will push the boundaries of rapidly developing technologies such as single particle cryo-EM, solid-state NMR and computational modeling as well as critically advance our understanding of molecular events that shape the formation of infectious virions.